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Abstract:

The present invention provides methods and devices for detecting a target
nucleic acid molecule. A set of oligonucleotide probes integrated into an
electric circuit, where the oligonucleotide probes are positioned such
that they cannot come into contact with one another, are contacted with a
sample. If the sample contains a target nucleic acid molecule, one which
has sequences complimentary to both probes, the target nucleic acid
molecule can bridge the gap between the probes. The resulting bridge can
then carry electrical current between the two probes, indicating the
presence of the target nucleic acid molecule.

Claims:

1. A device for detecting the presence of a target nucleic acid molecule,
comprising: two electronic leads, where an end of a first lead is located
near an end of second lead but where the leads are not in contact, one or
more sets of two oligonucleotide probes attached to the electronic leads,
where the oligonucleotide probes are positioned such that the probes can
not come into contact with one another and such that a target nucleic
acid molecule, which has two sequences, a first sequence complimentary to
a first probe attached to the first lead and a second sequence
complimentary to a second probe attached to the second lead, can bind to
both probes concurrently completing an electrical circuit, when the
target nucleic acid molecule is present, a fluidic channel for
introducing a reagent to coat the target nucleic acid molecule with a
metal conductor, an electric potential for generating a current flow
through the electrical circuit, a computer for detecting an electrical
current and correlating the presence or absence of an electrical current
to the presence or absence of the target nucleic acid molecule; wherein a
bridged probe having an electrical current flowing through the two
electrical leads and a target nucleic acid molecule denotes the presence
of the target nucleic acid molecule; and an unbridged probe lacks an
electrical current denoting the absence of the target nucleic acid
molecule.

2. The device according to claim 1, further comprising: a chamber for
treating a sample to release nucleic acid molecules from a sample.

3. The device according to claim 1, wherein the device contains proteins
for processing the sample.

4. The device according to claim 3, wherein the protein is selected from
the group consisting of a ligase, protease, restriction endonuclease,
nuclease, or nucleic acid binding protein.

5. The device according to claim 3, wherein the protein is a thermostable
protein.

6. The device according to claim 1, wherein the nucleic acid molecule is
DNA.

7. The device according to claim 1, wherein the nucleic acid molecule is
RNA.

8. The device according to claim 1, wherein the metal conductor is
silver.

9. The device according to claim 1, wherein the metal conductor is gold.

10. The device according to claim 1, further comprising: a chamber
containing nucleases for contacting the target nucleic acid after binding
with the probes.

11. The device according to claim 1, further comprising: heating elements
for heating the sample.

12. The device according to claim 1, wherein the probes are complimentary
to sequences from the genetic material of a pathogenic bacteria.

13. The device according to claim 12, wherein the pathogenic bacteria is
a biowarfare agent.

14. The device according to claim 12, wherein the pathogenic bacteria is
a food borne pathogen.

15. The device according to claim 1, wherein the probes are complimentary
to sequences from the genetic material of a virus.

16. The device according to claim 1, wherein the probes are complimentary
to sequences from the genetic material of a human.

17. The device according to claim 16, wherein one or both of the probes
has a sequence which is complimentary to a sequence having a
polymorphism, where the base or bases complimentary to the polymorphism
are located at the end of the probe.

18. A device for detecting the presence of a target nucleic acid
molecule, comprising: two electronic leads, where an end of a first lead
is located near an end of second lead but where the leads are not in
contact, one or more sets of two oligonucleotide probes attached to the
electronic leads, where the oligonucleotide probes are positioned such
that the probes can not come into contact with one another and such that
a target nucleic acid molecule, which has two sequences, a first sequence
complimentary to a first probe attached to the first lead and a second
sequence complimentary to a second probe attached to the second lead, can
bind to both probes concurrently completing an electrical circuit, when
the target nucleic acid molecule is present, a first microfluidic
channels for contacting the probes with a sample which may have the
target nucleic acid molecule to permit the target nucleic acid molecule,
if present in the sample, to bind to both probes, a second fluidic
channel for introducing a reagent to coat the target nucleic acid
molecule with a metal conductor, an electric potential for generating a
current flow through the electrical circuit, a computer for detecting an
electrical current, the electrical current indicating the presence of the
target nucleic acid molecule in the sample and no electrical current
indicating the lack of the target nucleic acid molecule in the sample.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 11/210,071
(filed Aug. 23, 2005) which is a continuation of U.S. Ser. No. 10/347,781
(filed Jan. 17, 2003), which is a divisional of U.S. Ser. No. 09/918,140
(filed Jul. 30, 2001), now U.S. Pat. No. 6,593,090, which is a
continuation of U.S. Ser. No. 09/545,010 (filed Apr. 7, 2000), now U.S.
Pat. No. 6,399,303, which claims benefit of U.S. Ser. No. 60/128,149
(filed Apr. 7, 1999). Each of these applications are hereby incorporated
by reference.

BACKGROUND OF THE INVENTION

[0002] DNA identification technology has numerous uses including
identification of pathogenic organisms, genetic testing, and forensics.
Advances have been made to allow for automated screening of thousands of
sequences concurrently. Gene chip technologies exist where DNA probes are
immobilized on a substrate such as a glass or silicon chip. A sample
containing nucleic acid molecules is applied to the chip and the nucleic
acid molecules within the sample are allowed to hybridize to the probe
DNA on the chip. Fluorescence detection is typically used to identify
double stranded nucleic acid molecule products. The advantage of the
system is the ability to screen hundreds or thousands of sequences using
automated systems.

[0003] Hybridization screening with fluorescence detection is a powerful
technique for detecting nucleic acid sequences. However, in order to
detect target DNA molecules, the target must first be amplified by PCR to
get a reliable signal. The gene chip technology also requires a system
capable of detecting fluorescent or radioactive materials. Such a system
is expensive to use and is not amenable to a portable system for
biological sample detection and identification. Furthermore, the
hybridization reactions take up to two hours. For many potential uses,
such as detecting biological warfare agents, the gene chip system is
simply not effective. Therefore, there is a need for a system which can
rapidly detect small quantities of a target nucleic acid molecule without
relying on PCR amplification.

[0004] The discussion above is merely provided for general background
information and is not intended to be used as an aid in determining the
scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE INVENTION

[0005] The present invention provides a method for detecting a target
nucleic acid molecule. A device for detecting the presence of a target
nucleic acid molecule is provided having two electronic leads, where the
ends of the leads are located near each other but are not in contact. One
or more sets of two oligonucleotide probes are attached to the electronic
leads. The oligonucleotide probes are positioned such that the probes can
not come into contact with one another and such that a target nucleic
acid molecule, which has two sequences complimentary to the probes can
bind to both probes concurrently. A sample which may have the target
nucleic acid molecule is contacted with the probes under selective
hybridization conditions. If the target is present it bridges the gap
between the probes. The target nucleic acid molecule may then carry
current between the probes, or be used as a support to form a conductive
wire between the two probes.

[0006] The present invention also provides a device for detecting the
presence of a target nucleic acid molecule. The device has two electronic
leads, where the ends of the leads are located near each other but are
not in contact. One or more sets of two oligonucleotide probes are
attached to the electronic leads. The oligonucleotide probes are
positioned such that the probes cannot come into contact with one another
and such that a target nucleic acid molecule, which has two sequences
complimentary to the probes can bind to both probes concurrently.

[0007] This brief description of the invention is intended only to provide
a brief overview of subject matter disclosed herein according to one or
more illustrative embodiments, and does not serve as a guide to
interpreting the claims or to define or limit the scope of the invention,
which is defined only by the appended claims. This brief description is
provided to introduce an illustrative selection of concepts in a
simplified form that are further described below in the detailed
description. This brief description is not intended to identify key
features or essential features of the claimed subject matter, nor is it
intended to be used as an aid in determining the scope of the claimed
subject matter. The claimed subject matter is not limited to
implementations that solve any or all disadvantages noted in the
background.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] So that the manner in which the features of the invention can be
understood, a detailed description of the invention may be had by
reference to certain embodiments, some of which are illustrated in the
accompanying drawings. It is to be noted, however, that the drawings
illustrate only certain embodiments of this invention and are therefore
not to be considered limiting of its scope, for the scope of the
invention encompasses other equally effective embodiments. The drawings
are not necessarily to scale, emphasis generally being placed upon
illustrating the features of certain embodiments of the invention. In the
drawings, like numerals are used to indicate like parts throughout the
various views. Thus, for further understanding of the invention,
reference can be made to the following detailed description, read in
connection with the drawings in which:

[0009] FIG. 1 graphically depicts the method of the present invention. Two
leads are provided each having a probe which is complimentary to
sequences on a target nucleic acid molecule (FIG. 1A). A target nucleic
acid molecule binds to the two probes at the complimentary sequences
(FIG. 1B). The complimentary strand is filled in (FIG. 1C). Nucleases are
used to remove the free ends of the target nucleic acid molecule (FIG.
1D). Current can be passed through the double stranded molecule or the
target nucleic acid molecule and probes may be coated with a conductor
and then tested for current flow.

[0010] FIG. 2 is a variation on the method shown in FIG. 1 using a ligase
method to distinguish a single base variation. The variation is
identified by the asterisk. After step D, a ligase is used. Only those
targets which have an exact match at the ends of the probes will ligate.
After ligation, the sample is heated to remove non-ligated target
molecules (FIG. 2E). The structure in FIG. 2E is stable at higher
temperatures, whereas the un-ligated structure in FIG. 2D would denature
under heat treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention provides devices and methods for rapidly
detecting the presence of nucleic acid molecules. The target nucleic acid
molecule either itself, or as a support, is used to complete a electrical
circuit. The presence of the target nucleic acid molecule is indicated by
the ability to conduct an electrical signal through the circuit. In the
case where the target nucleic acid molecule is not present, the circuit
is not be completed. Thus, the target nucleic acid molecule acts as a
switch. The presence of the nucleic acid molecule provides an on signal
for an electrical circuit, whereas the lack of the target nucleotide is
interpreted as an off signal. Due to the direct detection of the target
nucleic acid molecule, the method allows for extremely sensitive
detection of target molecules connect two wires.

[0012] The detection device is constructed on a support. Examples of
useful substrate materials include, e.g., glass, quartz and silicon as
well as polymeric substrates, e.g. plastics. In the case of conductive or
semi-conductive substrates, it will generally be desirable to include an
insulating layer on the substrate. However, any solid support which has a
non-conductive surface may be used to construct the device. The support
surface need not be flat. In fact, the support may be on the walls of a
chamber in a chip.

[0013] Two leads are provided having ends located close together, within
the spanning distance of a target nucleic acid molecule, but not
contacting one another. Current cannot flow effectively between the leads
without the presence of a target nucleic acid molecule to bridge the two
leads. Two probes specific to the target nucleic acid molecule are used.
The first is attached to one lead, the second to the other lead. The two
probes are specific to sequences on the target molecule which are
separated by sufficient distance to span the region between the leads.
Typically, the gap will be in micron or fractions of microns in length.
However, as chip manufacturing has improved, it has become possible to
shrink the distance between elements on a chip, requiring shorter lengths
of target nucleic acid molecules.

[0014] The target nucleic acid molecule is passed over the two leads. If a
target molecule has a sequence complimentary to one of the probes, it can
bind to that probe. Once bound to that probe, the nucleic acid molecule
is tethered at that site. The sequence complimentary to the second probe
can then bind to the second probe. To facilitate such a reaction, the two
complimentary sequences should be chosen such that the length of the
molecule in between can span the distance between the two leads and
provide flexibility for the nucleic acid molecule to move easily, such
that the second complimentary sequence readily binds to the second probe.

[0015] In a preferred embodiment, the probes are selected to bind with the
target such that they have approximately the same melting temperature.
This can be done by varying the lengths of the hybridization region. A-T
rich regions may have longer target sequences, whereas G-C rich regions
would have shorter target sequences.

[0016] Hybridization assays on substrate-bound oligonucleotide arrays
involve a hybridization step and a detection step. In the hybridization
step, a hybridization mixture containing the target and an isostabilizing
agent, denaturing agent or renaturation accelerant is brought into
contact with the probes of the array and incubated at a temperature and
for a time appropriate to allow hybridization between the target and any
complementary probes. Usually, unbound target molecules are then removed
from the array by washing with a wash mixture that does not contain the
target, such as hybridization buffer. This leaves only bound target
molecules. In the detection step, the probes to which the target has
hybridized are identified. In the present method the detection is carried
out by detecting a completed electronic circuit. Since the nucleotide
sequence of the probes at each feature is known, identifying the
locations at which target has bound provides information about the
particular sequences of these probes.

[0017] Including a hybridization optimizing agent in the hybridization
mixture significantly improves signal discrimination between perfectly
matched targets and single-base mismatches. As used herein, the term
"hybridization optimizing agent" refers to a composition that decreases
hybridization between mismatched nucleic acid molecules, i.e., nucleic
acid molecules whose sequences are not exactly complementary.

[0018] An isostabilizing agent is a composition that reduces the base-pair
composition dependence of DNA thermal melting transitions. More
particularly, the term refers to compounds that, in proper concentration,
result in a differential melting temperature of no more than about
1° C. for double stranded DNA oligonucleotides composed of AT or
GC, respectively. Isostabilizing agents preferably are used at a
concentration between 1 M and 10 M, between 2 M and 6 M, between 4 M and
6 M, between 4 M and 10 M and, optimally, at about 5 M. For example, 5 M
agent in 2×SSPE is suitable. Betaines and lower tetraalkyl ammonium
salts are examples of isostabilizing agents. In one embodiment, the
isostabilizing agent is not an alkylammonium ion.

[0019] Betaine (N,N,N,-trimethylglycine; (Rees et al., Biochem., (1993)
32:137-144), which is hereby incorporated by reference) can eliminate the
base pair composition dependence of DNA thermal stability. Unlike TMAC1,
betaine is zwitterionic at neutral pH and does not alter the
polyelectrolyte behavior of nucleic acids while it does alter the
composition-dependent stability of nucleic acids. Inclusion of betaine at
about 5 M can lower the average hybridization signal, but increases the
discrimination between matched and mismatched probes.

[0020] A denaturing agent is a compositions that lowers the melting
temperature of double stranded nucleic acid molecules by interfering with
hydrogen bonding between bases in a double-stranded nucleic acid or the
hydration of nucleic acid molecules. Denaturing agents can be included in
hybridization buffers at concentrations of about 1 M to about 6 M and,
preferably, about 3 M to about 5.5 M.

[0022] A renaturation accelerant is a compound that increases the speed of
renaturation of nucleic acids by at least 100-fold. They generally have
relatively unstructured polymeric domains that weakly associate with
nucleic acid molecules. Accelerants include heterogenous nuclear
ribonucleoprotein ("hnRP") A1 and cationic detergents such as,
preferably, CTAB ("cetyltrimethylammonium bromide") and DTAB ("dodecyl
trimethylammonium bromide"), and, also, polylysine, spermine, spermidine,
single stranded binding protein ("SSB"), phage T4 gene 32 protein and a
mixture of ammonium acetate and ethanol. Renaturation accelerants can be
included in hybridization mixtures at concentrations of about 1 mu M to
about 10 mM and, preferably, 1 mu M to about 1 mM. The CTAB buffers work
well at concentrations as low as 0.1 mM.

[0023] Homologous nucleotide sequences can be detected by selectively
hybridizing to each other. Selectively hybridizing is used herein to mean
hybridization of DNA or RNA probes from one sequence to the "homologous"
sequence under stringent or non-stringent conditions (Ausubel, et al.,
Eds., 1989, Current Protocols in Molecular Biology, Vol. I, Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, at
page 2.10.3, which is hereby incorporated by reference). Hybridization
and wash conditions are also exemplified in Sambrook, et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989), which is hereby incorporated by reference.

[0024] A variety of hybridization buffers are useful for the hybridization
assays of the invention. Addition of small amounts of ionic detergents
(such as N-lauroyl-sarkosine) are useful. LiCl is preferred to NaCl.
Hybridization can be at 20°-65° C., usually 37° C.
to 45° C. for probes of about 14 nucleotides. Additional examples
of hybridization conditions are provided in several sources, including:
Sambrook et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed.,
Cold Spring Harbor, N.Y.; and Berger and Kimmel, "Guide to Molecular
Cloning Techniques," Methods in Enzymology, (1987), Volume 152, Academic
Press, Inc., San Diego, Calif.; Young and Davis, Proc. Natl. Acad. Sci.
USA, 80:1194 (1983), which are hereby incorporated by reference.

[0025] In addition to aqueous buffers, non-aqueous buffers may also be
used. In particular non-aqueous buffers which facilitate hybridization
but have low electrical conductivity are preferred.

[0026] The hybridization mixture is placed in contact with the array and
incubated. Contact can take place in any suitable container, for example,
a dish or a cell specially designed to hold the probe array and to allow
introduction of the fluid into and removal of it from the cell so as to
contact the array. Generally, incubation will be at temperatures normally
used for hybridization of nucleic acids, for example, between about
20° C. and about 75° C., e.g., about 25° C., about
30° C., about 35° C., about 40° C., about 45°
C., about 50° C., about 55° C., about 60° C. or
about 65° C. For probes longer than about 14 nucleotides,
37° C.-45° C. is preferred. For shorter probes, 55°
C.-65° C. is preferred. More specific hybridization conditions can
be calculated using formulas for determining the melting point of the
hybridized region. Preferably, hybridization is carried out at a
temperature at or between ten degrees below the melting temperature and
the melting temperature. More preferred, the hybridization is carried out
at a temperature at or between five degrees below the melting temperature
and the melting temperature. The target is incubated with the probe array
for a time sufficient to allow the desired level of hybridization between
the target and any complementary probes in the array. After incubation
with the hybridization mixture, the array usually is washed with the
hybridization buffer, which also can include the hybridization optimizing
agent. These agents can be included in the same range of amounts as for
the hybridization step, or they can be eliminated altogether. Then the
array can be examined to identify the probes to which the target has
hybridized.

[0027] The target polynucleotide whose sequence is to be determined is
usually isolated from a tissue sample. If the target is genomic, the
sample may be from any tissue (except exclusively red blood cells). For
example, whole blood, peripheral blood lymphocytes or PBMC, skin, hair or
semen are convenient sources of clinical samples. These sources are also
suitable if the target is RNA. Blood and other body fluids are also a
convenient source for isolating viral nucleic acids. If the target is
mRNA, the sample is obtained from a tissue in which the mRNA is
expressed. If the polynucleotide in the sample is RNA, it may be reverse
transcribed to DNA, but in this method need not be converted to DNA.

[0028] Various methods exist for attaching the probes to the electronic
circuit. For example, U.S. Pat. Nos. 5,861,242; 5,861,242; 5,856,174;
5,856,101; and 5,837,832, which are hereby incorporated by reference,
disclose a method where light is shone through a mask to activate
functional (for oligonucleotides, typically an --OH) groups protected
with a photo-removable protecting group on a surface of a solid support.
After light activation, a nucleoside building block, itself protected
with a photo-removable protecting group (at the 5'-OH), is coupled to the
activated areas of the support. The process can be repeated, using
different masks or mask orientations and building blocks, to place probes
on a substrate.

[0030] Preferably, the probes are attached to the leads through spatially
directed oligonucleotide synthesis. Spatially directed oligonucleotide
synthesis may be carried out by any method of directing the synthesis of
an oligonucleotide to a specific location on a substrate. Methods for
spatially directed oligonucleotide synthesis include, without limitation,
light-directed oligonucleotide synthesis, microlithography, application
by ink jet, microchannel deposition to specific locations and
sequestration with physical barriers. In general these methods involve
generating active sites, usually by removing protective groups; and
coupling to the active site a nucleotide which, itself, optionally has a
protected active site if further nucleotide coupling is desired.

[0031] In one embodiment the lead-bound oligonucleotides are synthesized
at specific locations by light-directed oligonucleotide synthesis which
is disclosed in U.S. Pat. No. 5,143,854; PCT application WO 92/10092; and
PCT application WO 90/15070. In a basic strategy of this process, the
surface of a solid support modified with linkers and photolabile
protecting groups is illuminated through a photolithographic mask,
yielding reactive hydroxyl groups in the illuminated regions. A
3'-O-phosphoramidite-activated deoxynucleoside (protected at the
5'-hydroxyl with a photolabile group) is then presented to the surface
and coupling occurs at sites that were exposed to light. Following the
optional capping of unreacted active sites and oxidation, the substrate
is rinsed and the surface is illuminated through a second mask, to expose
additional hydroxyl groups for coupling to the linker. A second
5'-protected, 3'-O-phosphoramidite-activated deoxynucleoside (C-X) is
presented to the surface. The selective photodeprotection and coupling
cycles are repeated until the desired set of probes are obtained.
Photolabile groups are then optionally removed and the sequence is,
thereafter, optionally capped. Side chain protective groups, if present,
are also removed. Since photolithography is used, the process can be
miniaturized to specifically target leads in high densities on the
support.

[0032] This general process can be modified. For example, the nucleotides
can be natural nucleotides, chemically modified nucleotides or nucleotide
analogs, as long as they have activated hydroxyl groups compatible with
the linking chemistry. The protective groups can, themselves, be
photolabile. Alternatively, the protective groups can be labile under
certain chemical conditions, e.g., acid. In this example, the surface of
the solid support can contain a composition that generates acids upon
exposure to light. Thus, exposure of a region of the substrate to light
generates acids in that region that remove the protective groups in the
exposed region. Also, the synthesis method can use 3'-protected
5'-O-phosphoramidite-activated deoxynucleoside. In this case, the
oligonucleotide is synthesized in the 5' to 3' direction, which results
in a free 5' end.

[0033] The general process of removing protective groups by exposure to
light, coupling nucleotides (optionally competent for further coupling)
to the exposed active sites, and optionally capping unreacted sites is
referred to herein as "light-directed nucleotide coupling."

[0034] The probe molecules can be targeted to the leads through chemical
and electrical methods. The probes may be targeted to the leads by using
a chemical reaction for attaching the probe or nucleotide to the lead
which preferably binds the probe or nucleotide to the lead rather than
the support material. Alternatively, the probe or nucleotide may be
targeted to the lead by building up a charge on the lead which
electrostatically attracts the probe or nucleotide.

[0035] Nucleases can be used to remove probes which are attached to the
chip or lead in the wrong position. More particularly, a target nucleic
acid molecule may be added to the probes. Targets which bind at both ends
to probes, one end to each lead, will have no free ends and will be
resistant to exonuclease digestion. However, probes which are positioned
so that the target cannot contact both leads will be bound only one end,
leaving the molecule subject to digestion. Thus, improperly located
probes can be removed while protecting the properly located probes. After
the protease is removed or inactivated the target nucleic acid molecule
can be removed and the device is ready for use.

[0036] Interest has been growing in the fabrication of microfluidic
devices. Typically, advances in the semiconductor manufacturing arts have
been translated to the fabrication of micromechanical structures, e.g.,
micropumps, microvalves and the like, and microfluidic devices including
miniature chambers and flow passages.

[0037] A number of researchers have attempted employ these
microfabrication techniques in the miniaturization of some of the
processes involved in genetic analysis in particular. For example,
published PCT Application No. WO 94/05414, to Northrup and White,
incorporated herein by reference in its entirety for all purposes,
reports an integrated micro-PCR apparatus for collection and
amplification of nucleic acids from a specimen. U.S. Pat. No. 5,304,487
to Wilding et al., and U.S. Pat. No. 5,296,375 to Kricka et al., discuss
devices for collection and analysis of cell containing samples. Similar
techniques can be used to produce chips which can accept a sample,
release the nucleic acid molecules and then detect the target sequences.

[0038] Microfluidic devices are disclosed in U.S. Pat. No. 6,046,056,
which is hereby incorporated by reference. The devices includes a series
of channels fabricated into the surface of the substrate. At least one of
these channels will typically have very small cross sectional dimensions,
e.g., in the range of from about 0.1 micrometer to about 500 micrometers.
Preferably the cross-sectional dimensions of the channels will be in the
range of from about 0.1 to about 200 micrometers and more preferably in
the range of from about 0.1 to about 100 micrometers. In particularly
preferred aspects, each of the channels will have at least one
cross-sectional dimension in the range of from about 0.1 micrometers to
about 100 micrometers. Although generally shown as straight channels, it
will be appreciated that in order to maximize the use of space on a
substrate, serpentine, saw tooth or other channel geometries, to
incorporate effectively longer channels in shorter distances.

[0039] Manufacturing of these microscale elements into the surface of the
substrates may generally be carried out by any number of microfabrication
techniques that are well known in the art. For example, lithographic
techniques may be employed in fabricating, e.g., glass, quartz or silicon
substrates, using methods well known in the semi-conductor manufacturing
industries such as photolithographic etching, plasma etching or wet
chemical etching. Alternatively, micromachining methods such as laser
drilling, micromilling and the like may be employed.

[0040] Similarly, for polymeric substrates, well known manufacturing
techniques may also be used. These techniques include injection molding
or stamp molding methods where large numbers of substrates may be
produced using, e.g., rolling stamps to produce large sheets of
microscale substrates or polymer microcasting techniques where the
substrate is polymerized within a micromachined mold.

[0041] The devices will typically include an additional planar element
which overlays the channeled substrate enclosing and fluidly sealing the
various channels to form conduits. Attaching the planar cover element may
be achieved by a variety of means, including, e.g., thermal bonding,
adhesives or, in the case of certain substrates, e.g., glass, or
semi-rigid and non-rigid polymeric substrates, a natural adhesion between
the two components. The planar cover element may additionally be provided
with access ports and/or reservoirs for introducing the various fluid
elements needed for a particular screen.

[0042] The device may also include reservoirs disposed and fluidly
connected at the ends of the channels. A sample channel is used to
introduce the test compounds into the device. The introduction of a
number of individual, discrete volumes of compounds into the sample may
be carried out by a number of methods. For example, micropipettors may be
used to introduce the test compounds into the device. In preferred
aspects, an electropipettor may be used which is fluidly connected to
sample channel. Generally, an electropipettor utilizes electroosmotic
fluid direction, to alternately sample a number of test compounds, or
subject materials, and spacer compounds. The pipettor then delivers
individual, physically isolated samples into the sample channel for
subsequent manipulation within the device.

[0043] Alternatively, the sample channel may be individually fluidly
connected to a plurality of separate reservoirs via separate channels.
The separate reservoirs each contain a reactant compound, such as
proteins or detergents, with additional reservoirs being provided for
appropriate spacer compounds. The test compounds, reactant compounds,
and/or spacer compounds are then transported from the various reservoirs
into the sample channels using appropriate fluid direction schemes.

[0044] The sample collection portion of a device of the present invention,
whether or not on a micro scale, generally provides for the
identification of the sample, while preventing contamination of the
sample by external elements, or contamination of the environment by the
sample. Generally, this is carried out by introducing a sample for
analysis, e.g., preamplified sample, tissue, blood, saliva, etc.,
directly into a sample collection chamber within the device. Typically,
the prevention of cross-contamination of the sample may be accomplished
by directly injecting the sample into the sample collection chamber
through a sealable opening, e.g., an injection valve, or a septum.
Generally, sealable valves are preferred to reduce any potential threat
of leakage during or after sample injection. Alternatively, the device
may be provided with a hypodermic needle integrated within the device and
connected to the sample collection chamber, for direct acquisition of the
sample into the sample chamber. This can substantially reduce the
opportunity for contamination of the sample.

[0045] In addition to the foregoing, the sample collection portion of the
device may also include reagents and/or treatments for neutralization of
infectious agents, stabilization of the specimen or sample, pH
adjustments, and the like. Stabilization and pH adjustment treatments may
include, e.g., introduction of heparin to prevent clotting of blood
samples, addition of buffering agents, addition of protease or nuclease
inhibitors, preservatives and the like. Such reagents may generally be
stored within the sample collection chamber of the device or may be
stored within a separately accessible chamber, wherein the reagents may
be added to or mixed with the sample upon introduction of the sample into
the device. These reagents may be incorporated within the device in
either liquid or lyophilized form, depending upon the nature and
stability of the particular reagent used.

[0046] For those embodiments where whole cells, viruses or other tissue
samples are being analyzed, it will typically be necessary to extract the
nucleic acids from the cells or viruses, prior to continuing with the
various sample preparation operations. Accordingly, following sample
collection, nucleic acids may be liberated from the collected cells,
viral coat, etc., into a crude extract, followed by additional treatments
to prepare the sample for subsequent operations, e.g., denaturation of
contaminating (DNA binding) proteins, purification, filtration,
desalting, and the like.

[0047] Liberation of nucleic acids from the sample cells or viruses, and
denaturation of DNA binding proteins may generally be performed by
physical or chemical methods. For example, chemical methods generally
employ lysing agents to disrupt the cells and extract the nucleic acids
from the cells, followed by treatment of the extract with chaotropic
salts such as guanidinium isothiocyanate or urea to denature any
contaminating and potentially interfering proteins. Generally, where
chemical extraction and/or denaturation methods are used, the appropriate
reagents may be incorporated within the extraction chamber, a separate
accessible chamber or externally introduced.

[0048] Alternatively, physical methods may be used to extract the nucleic
acids and denature DNA binding proteins. U.S. Pat. No. 5,304,487, herein
incorporated by reference, discusses the use of physical protrusions
within microchannels or sharp edged particles within a chamber or channel
to pierce cell membranes and extract their contents. More traditional
methods of cell extraction may also be used, e.g., employing a channel
with restricted cross-sectional dimension which causes cell lysis when
the sample is passed through the channel with sufficient flow pressure.
Alternatively, cell extraction and denaturing of contaminating proteins
may be carried out by applying an alternating electrical current to the
sample. More specifically, the sample of cells is flowed through a
microtubular array while an alternating electric current is applied
across the fluid flow. A variety of other methods may be utilized within
the device of the present invention to effect cell lysis/extraction,
including, e.g., subjecting cells to ultrasonic agitation, or forcing
cells through microgeometry apertures, thereby subjecting the cells to
high shear stress resulting in rupture.

[0049] Following extraction, it will often be desirable to separate the
nucleic acids from other elements of the crude extract, e.g., denatured
proteins, cell membrane particles, and the like. Removal of particulate
matter is generally accomplished by filtration, flocculation or the like.
A variety of filter types may be readily incorporated into the device.
Further, where chemical denaturing methods are used, it may be desirable
to desalt the sample prior to proceeding to the next step. Desalting of
the sample, and isolation of the nucleic acid may generally be carried
out in a single step, e.g., by binding the nucleic acids to a solid phase
and washing away the contaminating salts or performing gel filtration
chromatography on the sample. Suitable solid supports for nucleic acid
binding include, e.g., diatomaceous earth, silica, or the like. Suitable
gel exclusion media is also well known in the art and is commercially
available from, e.g., Pharmacia and Sigma Chemical. This isolation and/or
gel filtration/desalting may be carried out in an additional chamber, or
alternatively, the particular chromatographic media may be incorporated
in a channel or fluid passage leading to a subsequent reaction chamber.

[0050] Alternatively, the interior surfaces of one or more fluid passages
or chambers may themselves be derivatized to provide functional groups
appropriate for the desired purification, e.g., charged groups, affinity
binding groups and the like.

[0051] In a preferred embodiment of the invention, ligation methods may be
used to specifically identify single base differences in sequences.
Previously, methods of identifying known target sequences by probe
ligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M.
Whiteley et al.; D. Y. Wu et al., Genomics, 4:560 (1989); U. Landegren et
al., Science, 241:1077 (1988); and E. Winn-Deen et al., Clin. Chem.,
37:1522 (1991), which are hereby incorporated by reference. In one
approach, known as oligonucleotide ligation assay ("OLA"), two probes or
probe elements which span a target region of interest are hybridized to
the target region. Where the probe elements basepair with adjacent target
bases, the confronting ends of the probe elements can be joined by
ligation, e.g., by treatment with ligase. The ligated probe element is
then assayed, evidencing the presence of the target sequence.

[0052] In the present invention, one or both probes may be designed to
specifically recognize a variation in the sequence at the end of the
probe. After the target binds to the probes, the target is treated with
nucleases to remove the ends of the molecules which do not bind to the
probes. The junction is then treated with ligase. If the complimentary
sequence is present at the end of the probe, the ligase will ligate the
target to the probe. The test chamber can then be heated up to denature
non-ligated targets. Detection of the specific target can then be carried
out.

[0053] In one embodiment of the invention, the probe set is contacted with
a target nucleic acid molecule and after hybridization the nucleic acid
molecules are coated with a conductor, such as a metal, as described in
U.S. Patent Applications Ser. Nos. 60/095,096, 60/099,506, or Ser. No.
09/315,750 which are hereby incorporated by reference. The coated nucleic
acid molecule can then conduct electricity across the gap between the
pair of probes, thus producing a detectable signal indicative of the
presence of a target nucleic acid molecule.

[0054] Braun demonstrated that silver could be deposited along a DNA
molecule. A three-step process is used. First, silver is selectively
localized to the DNA molecule through a Ag+/Na+ ion-exchange (Barton, in
Bioinorganic Chemistry (eds Bertini, et al.) ch. 8 (University Science
Books, Mill Valley, 1994, which is hereby incorporated by reference) and
complexes are formed between the silver and the DNA bases (Spiro (ed.)
Nucleic Acid-Metal Ion Interactions (Wiley Interscience, New York 1980;
Marzeilli, et al., J. Am. Chem. Soc. 99:2797 (1977); Eichorn (ed.)
Inorganic Biochemistry, Vol. 2, ch 33-34 (Elsevier, Amsterdam, 1973),
which are hereby incorporated by reference). The ion-exchange process may
be monitored by following the quenching of the fluorescence signal of the
labeled DNA. The silver ion-exchanged DNA is then reduced to form
aggregates with bound to the DNA skeleton. The silver aggregates are
further developed using standard procedures, such as those used in
photographic chemistry (Holgate, et al., J. Histochem. Cytochem. 31:938
(1983); Birell, et al., J. Histochem. Cytochem. 34:339 (1986), which are
hereby incorporated by reference).

[0055] The nucleic acid molecule itself may have some conductive
properties of its own. These properties may be modified to reduce any
detrimental effects on the function of the electronic circuit (Meade, et
al, U.S. Pat. No. 5,770,369, "Nucleic Acid Mediated Electron Transfer"
(1998), which is hereby incorporated by reference). Modification of the
electrical properties of the nucleic acid molecule may be made by
intercalating compounds between the bases of the nucleic acid molecule,
modifying the sugar-phosphate backbone, or by cleaving the nucleic acid
molecule after the circuit elements are formed. Cleavage of the nucleic
acid molecule may be accomplished by irradiation, chemical treatment, or
enzymatic degradation. Irradiation using gamma-radiation is preferred
because radiation may penetrate materials coating the nucleic acid
molecule.

[0056] In another aspect of the invention, the electrical conductivity of
nucleic acid molecules is relied upon to transmit the electrical signal.
Hans-Werner Fink and Christian Schoenenberger reported in Nature (1999),
which is hereby incorporated by reference, that double-stranded DNA
conducts electricity like a semiconductor. This flow of current can be
sufficient to construct a simple switch. The present invention provides
an electronic detector based upon such a nucleic acid switch, which will
indicate whether or not a target nucleic acid molecule is present within
a sample.

[0057] Probes to the target nucleic acid molecule are immobilized within
an electrical circuit. The probes are physically located at a distance
sufficient that they cannot come into contact with one another. The
sample to be tested is contacted with the probes. If a nucleic acid
molecule is present in the sample which has sequences homologous or
complementary to the two probes, the nucleic acid molecule can bridge the
gap between the probes. The detection unit can then detect an electrical
current which can flow through the nucleic acid molecule. A computer unit
can detect the presence of the nucleic acid molecule as an "on" switch,
while an unbridged probe set would be an "off" switch. The information is
processed by a digital computer which correlates the status of the switch
with the presence of a particular target. The computer can also analyze
the results from several switches specific for the same target, to
determine specificity of binding and target concentration. The
information could be quickly identified to the user by indicating the
presence or absence of the biological material, organism, mutation, or
other target of interest on the nucleic acid molecule.

[0058] A detection device could comprise numerous different probe sets
which could detect a wide variety of targets. Thus a detection device
could screen for multiple target DNA molecules. For example, a detection
device could have probe sets directed at multiple pathogenic organisms.
In that way, a sample could be screened for several pathogens
simultaneously. Each probe set would be a separate switch which would
indicate the presence or absence of the complimentary nucleic acid
molecule.

[0059] A cell sample can be prepared by either chemical (including
enzymatic) or physical disruption, or a combination thereof. After lysis
the sample can be further processed. For example, the sample can be
treated with RNase to remove any RNA to limit detection to DNA.

[0060] Prior to or at the point of contact with the probes, the nucleic
acid molecules in the sample are denatured. Denaturation is
preferentially carried out by heat treatment. Denaturation can also be
carried out by varying the ionic concentration of the carrier solution or
by a combination of ionic and heat treatment.

[0061] The present invention also has the advantage of being used for
multiple samples. The probe sets can be recycled by stripping the target
DNAs from the probe sets. In a preferred embodiment the stripping is
accomplished by increasing temperature and/or salt concentration. The
probe set is then ready for analysis of an additional sample.

[0063] In one embodiment, the bridging nucleic acid molecule can be made
double stranded by adding a segment of a nucleic acid molecule which is
complimentary to the region of the target nucleic acid molecule located
between the sequences complimentary to the probes. Ligase can be used to
ligate the fragments into one molecule. The device may be recycled by
passing through a restriction endonuclease to release the bridging
nucleic acid molecule. Alternatively, a polymerase can be used to fill in
the complimentary sequence. In that case, the solution must contain
nucleotides for the synthesis of the complimentary strand.

[0064] Each probe set consists of two probes. Each probe may consist of
one or more copies of the oligonucleotide, where all the copies for that
probe attach to the circuit so that electrical current can be carried
through the probe and to the circuit. A connection between any of the
oligonucleotides in one probe with any of the oligonucleotides in the
other probe of the set will complete the circuit producing an "on"
signal. If the probes consist of multiple copies of the oligonucleotides
and/or if multiple probes are used, the device can be used to quantitate
the level of the target nucleic acid molecule in the sample, by the
signal strength or the number of activated switches.

[0065] The number of probes may be increased in order to determine
concentrations of the target nucleic acid molecule. For example, several
thousand repeated probes may be produced in the detection unit. The
circuit would be able to count the number of occupied sites. Calculations
could be done by the unit to determine the concentration of the target
molecule.

[0066] The present invention can be used for numerous applications, such
as detection of pathogens. For example, samples may be isolated from
drinking water or food and rapidly screened for infectious organisms.
This invention may also be used for DNA sequencing using hybridization
techniques. Such methods are described in U.S. Pat. No. 5,837,832, which
is hereby incorporated by reference. The present invention may be used to
screen for mutations or polymorphisms in samples isolated from patients.

[0067] The present invention may also be used for food and water testing.
In recent times, there have been several large recalls of tainted meat
products. The electronic DNA detection system can be used for the
in-process detection of pathogens in foods and the subsequent disposal of
the contaminated materials. This could significantly improve food safety,
prevent food borne illnesses and death, and avoid costly recalls. Chips
with probes that can identify common food borne pathogens, such as
Salmonella and E. Coli, could be designed for use within the food
industry.

[0068] In yet another embodiment, the present invention can be used for
real time detection of biological warfare agents: With the recent
concerns of the use of biological weapons in a theater of war and in
terrorist attacks, the device could be configured into a personal sensor
for the combat soldier or into a remote sensor for advanced warnings of a
biological threat. The devices which can be used to specifically identity
of the agent, can be coupled with a modem to send the information to
another location. Mobile devices may also include a global positioning
system to provide both location and pathogen information.

[0069] In yet another embodiment, the present invention may be used to
identify an individual. A series of probes, of sufficient number to
distinguish individuals with a high degree of reliability, are placed
within the device. Various polymorphism sites are used. Preferentially,
the device can determine the identity to a specificity of greater than
one in I million, more preferred is a specificity of greater than one in
one billion, even more preferred is a specificity of greater than one in
ten billion.

[0070] As an example, a flow chart is provided indicating how a cell
sample can be tested for the presence of a target nucleic acid molecule:

[0071] 1. Inject sample

[0072] 2. Lyse cells

[0073] 3. Process lysate

[0074] 4. Denature nucleic acid molecules

[0075] 4. Contact sample with probe sets--under stringent conditions

[0076] 5. Determine whether current can travel between a probe set

[0077] 6. Correlate the current signal with a positive identification of
the target DNA

[0078] Note that not all steps are required depending upon the
application. For example, lysis is only needed if the DNA is still within
a cell.

[0079] Control probe sets can be utilized to verify that the system is
working appropriately. The probe sets can recognize sequences known to
occur within the sample or be nucleic acid molecules which are added to
the sample.

[0080] Controls are especially useful to determine the presence of
sequences having a polymorphism. Control nucleic acid molecules lacking
the polymorphism may be compared in a separate test. In a preferred
embodiment, the control sequence is tested at the same time in a separate
chamber in the device.

[0081] The correct control sequence will hybridize to the probes at a
slightly higher temperature. This difference can be used to differentiate
the single base mutant from the correct sequence. The device will
indicate binding by the correct sequence at a temperature where the
mutant sequence cannot bind. However, at a lower temperature, both
sequences will bind.

[0082] In yet another embodiment, the nucleotide probes on the substrate
may be randomly chosen. A linker nucleic acid molecule comprising a
complimentary sequence to the substrate bound probe and a sequence
complimentary to the target nucleic acid molecule (See FIG. 2). Thus the
linker can be used to make the probe sequence able to detect any target
nucleic acid sequence without having to modify the device itself. Rather
the linker molecule may be bound to the substrate bound nucleic acid
molecule either before or together with the sample to be tested. If
desired the linker may be ligated to the substrate bound probe. This
would allow for the reuse of the linker with multiple samples.

[0083] The present invention can be used to monitor gene expression in
cells. The level of RNA is determined using multiple switches with probes
complimentary to the target RNA molecule. Samples can be taken at various
times after a stimulus or at different stages of development.

[0084] In yet another embodiment, the present invention can be used to
sequence nucleic acid molecules. Sequencing by hybridization (SBH) is
most efficiently practiced by attaching many probes to a surface to form
an array in which the identity of the probe at each site is known. A
labeled target DNA or RNA is then hybridized to the array, and the
hybridization pattern is examined to determine the identity of all
complementary probes in the array. Contrary to the teachings of the prior
art, which teaches that mismatched probe/target complexes are not of
interest, the present invention provides an analytical method in which
the hybridization signal of mismatched probe/target complexes identifies
or confirms the identity of the perfectly matched probe/target complexes
on the array.

[0085] Techniques for sequencing a nucleic acid using a probe array have
been disclosed in PCT Application No. 92/10588, which is hereby
incorporated by reference. Each probe is located at a positionally
distinguishable location on the substrate. When the labeled target is
exposed to the substrate, it binds at locations that contain
complementary nucleotide sequences. Through knowledge of the sequence of
the probes at the binding locations, one can determine the nucleotide
sequence of the target nucleic acid. The technique is particularly
efficient when very large arrays of nucleic acid probes are utilized.

[0086] In a preferred embodiment, the device consists of a detection chip
having the microfluidic structures needed to release the nucleic acid
molecules from a sample. The nucleic acid molecules are introduced into a
chamber with the detection system having the probes. The detection
switches are connected to a processor which can analyze the results from
the hybridization reactions. A user interface, such as a screen is
provided for the user to read the results. In addition, the device may
have additional information in memory or accessible by modem regarding
the organism or individual from which the target nucleic acid molecule
was derived.

EXAMPLES

Example 1

Preparation of a Sample to Detect Pathogens

[0087] A sample to be tested is isolated. A common sample would be a blood
sample from a patient. The sample is injected into the device. The sample
moves into a chamber where it is treated chemically, with detergents, and
enzymatically, with proteases to free nucleic acid molecules from cells
in the sample. Heat treatment is also used to facilitate the release of
the nucleic acid molecules. For that reason, proteins used in the present
invention are preferably thermostable. The mixture may then pass though a
filter on the chip to partially purify the nucleic acid molecules.

Example 2

Preparation of Oligonucleotide Probe Sets

[0088] Each oligonucleotide probe set is selected so that the two probes
are complimentary to a portion of the target nucleic acid molecule and so
that the two portions of the target nucleic acid molecule are located
sufficiently far apart that the nucleic acid molecule can bridge the gap
between the two probes on the device when they are both bound. The
complimentary sequences will be chosen such that there is some additional
length to allow the target nucleic acid molecule to move freely when
bound by one probe, so that it may access the second probe. Preferably
the molecule will not be much longer than needed to easily bridge the
gap. As the length of the molecule increases the chance of it locating
the second probe decreases, because the effective concentration of the
binding site on the target molecule decreases as the volume in which it
can move increases.

[0089] Each probe set will be attached to a substrate so that they are
positioned as discussed above.

Example 3

Testing for the Presence of the Target Nucleic Acid Molecule

[0090] The probe sets will be contacted with the nucleic acid molecules.
The test chamber has a small volume to facilitate binding of the target
to the probe. To increase the chance of binding, the sample is circulated
multiple times through the test chamber. The sample will flow through a
test chamber containing the probe sets, at a flow rate sufficiently low
to allow the target nucleic acid molecules to bind to a probe. Conditions
are determined by the length and sequence of the probe.

[0091] The conditions will be set at a level where the stringency is
sufficient to eliminate non-specific binding to the probes. The target
nucleic acid molecule is contacted with the probes under stringent
conditions. The stringent conditions for hybridization are by the nucleic
acid, salt, and temperature. These conditions are well known in the art
and may be-altered in order to identify or detect identical or related
polynucleotide sequences. Numerous equivalent conditions comprising
either low or high stringency depend on factors such as the length and
nature of the sequence (DNA, RNA, base composition), nature of the target
(DNA, RNA, base composition), milieu (in solution or immobilized on a
solid substrate), concentration of salts and other components (e.g.,
formamide, dextran sulfate and/or polyethylene glycol), and temperature
of the reactions. One or more factors be may be varied to generate
conditions of either low or high stringency different from, but
equivalent to, the above listed conditions.

[0092] The test chamber is then rinsed with a solution to remove unbound
nucleic acid molecules. A solution which is non-conducting lowers the
level of false positives by cutting down on conductivity mediated by the
buffer.

[0093] A current is then applied at one lead while a detector looks for a
signal at the other lead. A current between the two leads is indicative
of the presence of the target nucleic acid molecule.

[0094] This written description uses examples to disclose the invention,
including the best mode, and also to enable any person skilled in the art
to practice the invention, including making and using any devices or
systems and performing any incorporated methods. The patentable scope of
the invention is defined by the claims, and may include other examples
that occur to those skilled in the art. Such other examples are intended
to be within the scope of the claims if they have structural elements
that do not differ from the literal language of the claims, or if they
include equivalent structural elements with insubstantial differences
from the literal language of the claims.

Patent applications in class Nucleotides or polynucleotides, or derivatives thereof

Patent applications in all subclasses Nucleotides or polynucleotides, or derivatives thereof